T cell interactions with B cells during germinal center formation,a three‐step model

Establishment of effective immunity against invading microbes depends on continuous generation of antibodies that facilitate pathogen clearance. Long‐lived plasma cells with the capacity to produce high affinity antibodies evolve in germinal centers (GCs), where B cells undergo somatic hypermutation and are subjected to affinity‐based selection. Here, we focus on the cellular interactions that take place early in the antibody immune response during GC colonization. Clones bearing B‐cell receptors with different affinities and specificities compete for entry to the GC, at the boundary between the B‐cell and T‐cell zones in lymphoid organs. During this process, B cells compete for interactions with T follicular helper cells, which provide selection signals required for differentiation into GC cells and antibody secreting cells. These cellular engagements are long‐lasting and depend on activation of adhesion molecules that support persistent interactions and promote transmission of signals between the cells. Here, we discuss how interactions between cognate T and B cells are primarily maintained by three types of molecular interactions: homophilic signaling lymphocytic activation molecule (SLAM) interactions, T‐cell receptor: peptide‐loaded major histocompatibility class II (pMHCII), and LFA‐1:ICAMs. These essential components support a three‐step process that controls clonal selection for entry into the antibody affinity maturation response in the GC, and establishment of long‐lasting antibody‐mediated immunity.     

BAFF inhibition in SLE—Is tolerance restored?

The B cell activating factor (BAFF) inhibitor, belimumab, is the first biologic drug approved for the treatment of SLE, and exhibits modest, but durable, efficacy in decreasing disease flares and organ damage. BAFF and its homolog APRIL are TNF‐like cytokines that support the survival and differentiation of B cells at distinct developmental stages. BAFF is a crucial survival factor for transitional and mature B cells that acts as rheostat for the maturation of low‐affinity autoreactive cells. In addition, BAFF augments innate B cell responses via complex interactions with the B cell receptor (BCR) and Toll like receptor (TLR) pathways. In this manner, BAFF impacts autoreactive B cell activation via extrafollicular pathways and fine tunes affinity selection within germinal centers (GC). Finally, BAFF and APRIL support plasma cell survival, with differential impacts on IgM‐ and IgG‐producing populations. Therapeutically, BAFF and combined BAFF/APRIL inhibition delays disease onset in diverse murine lupus strains, although responsiveness to BAFF inhibition is model dependent, in keeping with heterogeneity in clinical responses to belimumab treatment in humans. In this review, we discuss the mechanisms whereby BAFF/APRIL signals promote autoreactive B cell activation, discuss whether altered selection accounts for therapeutic benefits of BAFF inhibition, and address whether new insights into BAFF/APRIL family complexity can be exploited to improve human lupus treatments.     

SHIP recruitment attenuates Fc gamma RIIB-induced B cell apoptosis.

Fc gammaRIIB is an inhibitory receptor that terminates activation signals initiated by antigen cross-linking of the BCR through the recruitment of SHIP. Fc gammaRIIB can also signal independently of BCR coligation to directly mediate an apoptotic response, requiring only an intact transmembrane domain. Failure to recruit SHIP, either by deletion of SHIP or mutation of Fc gammaRIIB, results in enhanced Fc gammaRIIB-triggered apoptosis. Thus, in the germinal center, where ICs are retained by FDCs, Fc gammaRIIB may be an active determinant in the negative selection of B cells whose BCRs have reduced affinity for antigen as a result of somatic hypermutation. Selection of B cells may represent the sum of opposing signals generated by the interaction of ICs with the BCR and Fc gammaRIIB through pathways modulated by SHIP.     

Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies

The humoral immune response requires antigen-specific B cell activation and subsequent terminal differentiation into plasma cells. Engagement of B cell antigen receptor (BCR) on mature B cells activates an intracellular signaling cascade, including calcium mobilization, which leads to cell proliferation and differentiation. Coengagement by immune complex of BCR with the inhibitory Fc receptor FcgammaRIIb, the only IgG receptor expressed on B cells, inhibits B cell activation signals through a negative feedback loop. We now describe antibodies that mimic the inhibitory effects of immune complex by high-affinity coengagement of FcgammaRIIb and the BCR coreceptor complex on human B cells. We engineered the Fc domain of an anti-CD19 antibody to generate variants with up to approximately 430-fold greater affinity to FcgammaRIIb. Relative to native IgG1, the FcgammaRIIb binding-enhanced (IIbE) variants strongly inhibited BCR-induced calcium mobilization and viability in primary human B cells. Inhibitory effects involved phosphorylation of SH2-containing inositol polyphosphate 5-phosphatase (SHIP), which is known to be involved in FcgammaRIIb-induced negative feedback of B cell activation by immune complex. Coengagement of BCR and FcgammaRIIb by IIbE variants also overcame the anti-apoptotic effects of BCR activation. The use of a single antibody to suppress B cell functions by coengagement of BCR and FcgammaRIIb may represent a novel approach in the treatment of B cell-mediated autoimmune diseases.     

B‐cell‐intrinsic function of TAPP adaptors in controlling germinal center responses and autoantibody production in mice

Control of B‐cell signal transduction is critical to prevent production of pathological autoantibodies. Tandem PH domain containing proteins (TAPPs) specifically bind PI(3,4)P2, a phosphoinositide product generated by PI 3‐kinases and the phosphatase SHIP. TAPP KI mice bearing PH domain‐inactivating mutations in both TAPP1 and TAPP2 genes, uncoupling them from PI(3,4)P2, exhibit increased BCR‐induced activation of the kinase Akt and develop lupus‐like characteristics including anti‐DNA antibodies and deposition of immune complexes in kidneys. Here, we find that TAPP KI mice develop chronic germinal centers (GCs) with age and show abnormal expression of B‐cell activation and memory markers. Upon immunization with T‐dependent Ag, TAPP KI mice develop functional but abnormally large GCs, associated with increased GC B‐cell survival. Disruption of chronic GCs in TAPP KI mice by deletion of the costimulatory molecule ICOS abrogate anti‐DNA and anti‐nuclear antibody production in TAPP KI mice, indicating an essential role for GCs. Moreover, TAPP KI B cells are sufficient to drive chronic GC responses and recapitulate the autoimmune phenotype in BM chimeric mice. Our findings demonstrate a B‐cell‐intrinsic role of TAPP–PI(3,4)P2 interaction in regulating GC responses and autoantibody production and suggest that uncontrolled Akt activity in B cells can drive autoimmunity.     

Regulation of immune cell signaling by SHIP1: A phosphatase,scaffold protein,and potential therapeutic target

The phosphoinositide phosphatase SHIP is a critical regulator of immune cell activation. Despite considerable study, the mechanisms controlling SHIP activity to ensure balanced cell activation remain incompletely understood. SHIP dampens BCR signaling in part through its association with the inhibitory coreceptor Fc gamma receptor IIB, and serves as an effector for other inhibitory receptors in various immune cell types. The established paradigm emphasizes SHIP's inhibitory receptor‐dependent function in regulating phosphoinositide 3‐kinase signaling by dephosphorylating the phosphoinositide PI(3,4,5)P₃; however, substantial evidence indicates that SHIP can be activated independently of inhibitory receptors and can function as an intrinsic brake on activation signaling. Here, we integrate historical and recent reports addressing the regulation and function of SHIP in immune cells, which together indicate that SHIP acts as a multifunctional protein controlled by multiple regulatory inputs, and influences downstream signaling via both phosphatase‐dependent and ‐independent means. We further summarize accumulated evidence regarding the functions of SHIP in B cells, T cells, NK cells, dendritic cells, mast cells, and macrophages, and data suggesting defective expression or activity of SHIP in autoimmune and malignant disorders. Lastly, we discuss the biological activities, therapeutic promise, and limitations of small molecule modulators of SHIP enzymatic activity.     

Monomer hapten and hapten‐specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co‐ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2‐containing inositol 5’‐phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP‐alanine) to multivalent antigen (TNP‐OVA)‐activated rat basophilic leukemia cells and mouse bone marrow‐derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP‐OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca²⁺ concentrations ([Ca²⁺]_i), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten‐specific IgG monoclonal antibody (anti‐TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co‐ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca²⁺]_i, weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.     

The SH2 domain containing inositol 5-phosphatase SHIP2 associates to the immunoreceptor tyrosine-based inhibition motif of Fc gammaRIIB in B cells under negative signaling

Fc gammaRIIB are single-chain low-affinity receptors for IgG that bear an immunoreceptor tyrosine-based inhibition motif (ITIM) in their intracytoplasmic domain and that negatively regulate immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. In B cells, coaggregation of the B cell receptor (BCR) and Fc gammaRIIB leads to an inhibition of B cell activation. Inhibitory properties of Fc gammaRIIB have been related to the recruitment of SHIP, an SH2 domain-containing inositol 5-phosphatase (referred to as SHIP1), via ITIM phosphorylated Fc gammaRIIB. Here, we demonstrate that the second SH2 domain-containing inositol 5-phosphatase SHIP2 could also bind to the Fc gammaRIIB ITIM. As a model, a Fc gammaRIIB deficient B cell line (IIA1.6), transfected with a cDNA encoding either w.t. Fc gammaRIIB1' or Fc gammaRIIB1' whose ITIM tyrosine was mutated has been used. SHIP2 tyrosine phosphorylation and association to the adaptator protein Shc were only found in transfectants expressing w.t. Fc gammaRIIB1'. SHIP2 was also found to bind to a phosphopeptide corresponding to the ITIM sequence of Fc gammaRIIB. There was no binding to the nonphosphorylated peptide. Finally, both SHIP2 and SHIP1 were coprecipitated with Fc gammaRIIB1' upon coaggregation with BCR in IIA1.6 transfectants.     

Con A activates an Akt/PKB dependent survival mechanism to modulate TCR induced cell death in double positive thymocytes

While low avidity ligation of the T cell receptor (TCR) leads to positive selection and further maturation of developing thymocytes providing the immune system with mature CD4(+) and CD8(+) (single positive) T cells, high avidity ligation triggers negative selection by apoptotic cell death and therefore the TCR repertoire is purged of autoreactive T cells. On peripheral T cells, however, high avidity ligation of the TCR triggers activation and survival not death. In the present study we used concanavalin A (Con A) and alpha-CD3 epsilon antibody to investigate a possible survival mechanism in connection with TCR ligation. Con A and alpha-CD3 epsilon were used in the study for the following reasons: (1) they both mimic the effects of high avidity TCR ligation by activating peripheral T cells, and (2) they trigger distinctively different physiological changes in developing thymocytes. While Con A supports events associated with cellular survival, alpha-CD3 epsilon induces apoptotic cell death. In our experimental system the TCR was cross-linked by Con A and alpha-CD3 epsilon in thymocytes of major histocompatibility complex (MHC) deficient thymus organ cultures, where signals from the TCR can be triggered on zero background signal level. We have found that TCR cross-linking by Con A and not by alpha-CD3 epsilon decreases the gene and protein expression of the pro-apoptotic molecule, Bad; and that Con A is capable of the activation of the survival signalling pathway including protein kinase B (Akt/PKB) independently of phosphatidyl inositol kinase (PI3K).     

Basal B-cell receptor signaling in B lymphocytes: mechanisms of regulation and role in positive selection, differentiation, and peripheral survival

Summary:  B-cell development is a highly ordered multistep process dependent upon signals generated by the pre-B and B-cell antigen receptor (BCR). BCR signals drive maturation of the B cell by integrating a number of parallel and sequential biological processes that result in generation of fully immunocompetent B cells. Among these biological processes are positive selection through several developmental checkpoints, negative selection of potentially self-reactive B cells, and activation of the mature B cell. In addition, recent studies have shown that developing and mature B cells rely on the constant activity of the BCR for their continued survival. Ligand (antigen)-dependent and -independent mechanisms of BCR signaling have been proposed, but their specific contributions to B-cell maturation and differentiation in the bone marrow and periphery are not completely clear. We discuss here a model, whereby ligand-independent basal BCR activity would be sufficient to trigger B-cell development through to the mature stage. However, long-term survival and formation of specific mature B-cell populations may be dependent on ligand–receptor interactions.     

Selective regulation of autoreactive B cells by FcγRIIB

FcγRIIB is an inhibitory receptor which plays a role in limiting B cell and DC activation. Since FcγRIIB is known to dampen the signaling strength of the BCR, we wished to determine the impact of FcγRIIB on the regulation of BCRs which differ in their affinity for DNA. For these studies, FcγRIIB deficient BALB/c mice were bred with mice expressing the transgene-encoded H chain of the R4A anti-DNA antibody which gives rise to BCRs which express high, low or no affinity for DNA. The deletion of FcγRIIB in R4A BALB/c mice led to an alteration in the B cell repertoire, allowing for the expansion and activation of high affinity DNA-reactive B cells. By 6–8 months of age, R4A × FcγRIIB^−/− BALB/c mice spontaneously developed anti-DNA antibody titers. These mice also displayed an induction of IFN-inducible genes and an elevation in levels of the B cell survival factor, BAFF. These data demonstrate that FcγRIIB preferentially limits activation of high affinity autoreactive B cells and can influence the activation of DC through an immune complex-mediated mechanism.     

An analysis of B cell selection mechanisms in germinal centers.

Affinity maturation of antibodies during immune responses is achieved by multiple rounds of somatic hypermutation and subsequent preferential selection of those B cells that express B cell receptors with improved binding characteristics for the antigen. The mechanism underlying B cell selection has not yet been defined. By employing an agent-based model, we show that for physiologically reasonable parameter values affinity maturation can be driven by competition for neither binding sites nor antigen--even in the presence of competing secreted antibodies. Within the tested mechanisms, only clonal competition for T cell help or a refractory time for the interaction of centrocytes with follicular dendritic cells is found to enable affinity maturation while generating the experimentally observed germinal centre characteristics and tolerating large variations in the initial antigen density.     

NCR1‐deficiency diminishes the generation of protective murine cytomegalovirus antibodies by limiting follicular helper T‐cell maturation

NKp46/NCR1 is an activating NK‐cell receptor implicated in the control of various viral and bacterial infections. Recent findings also suggest that it plays a role in shaping the adaptive immune response to pathogens. Using NCR1‐deficient (NCR1^gfp/gfp) mice, we provide evidence for the role of NCR1 in antibody response to mouse cytomegalovirus infection (MCMV). The absence of NCR1 resulted in impaired maturation, function and NK‐cell migration to regional lymph nodes. In addition, CD4⁺ T‐cell activation and follicular helper T‐cell (Tfh) generation were reduced, leading to inferior germinal center (GC) B‐cell maturation. As a consequence, NCR1^gfp/gfp mice produced lower amounts of MCMV‐specific antibodies upon infection, which correlated with lower number of virus‐specific antibody secreting cells in analyzed lymph nodes.     

Complement C4 maintains peripheral B‐cell tolerance in a myeloid cell dependent manner

The factors that allow self‐reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock‐in mouse strain, we identify a pathway by which B‐cell selection to nucleolar self‐antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B‐cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self‐reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self‐reactive B‐cell clones leading to increased spontaneous formation of GCs.     

Predominant neuronal B-cell loss in L5 DRG of p75 receptor-deficient mice

The significance of the p75 low‐affinity neurotrophin receptor, for the maintenance and survival of DRG cells, was studied in p75‐deficient mice. Perikarya of the L5 DRG of 12‐week‐old p75 receptor‐deficient mice and healthy Balb C mice were compared using stereological techniques. Following systematic sampling, the optical fractionator and the planar vertical rotator were used to estimate the number and mean volume of the cell bodies of the two neuronal subpopulations. The loss of B‐cells was 57% (P < 0.00001), numbers being 7300 (CV = 0.12) in controls and 3100 in p75 receptor‐deficient mice (CV = 0.18). Also, A‐cells showed a significant loss of 39% (P < 0.0001), numbers being 2600 (CV = 0.12) in control mice and 1500 (CV = 0.16) in p75 receptor‐deficient mice. The volume of A‐cells was reduced by 30% (P < 0.01), from 24.700 µm³ (CV = 0.17) perikarya in p75 knock‐out mice to 15.100 µm³ (CV = 0.17) in controls. B‐cell volume did not change significantly. It is concluded that the p75 receptor plays a major role in the survival of DRG cells. The predominant loss of small B‐cells indicates that the effect of neurotrophins is dependent upon the presence of the p75 low‐affinity receptor.     

TAK1 maintains the survival of immunoglobulin λ‐chain‐positive B cells

TAK1 (MAP3K7) mediation of the IκB kinase (IKK) complex?nuclear factor‐κB (NF‐κB) pathway is crucial for the activation of immune response and to perpetuate inflammation. Although progress has been made to understand TAK1 function in the B‐cell receptor (BCR) signaling, the physiological roles of TAK1 in B‐cell development, particularly in the bone marrow (BM), remain elusive. Previous studies suggested that the IKK complex is required for the development of immunoglobulin light chain λ‐positive B cells, but not for receptor editing. In contrast, NF‐κB activity is suggested to be involved in the regulation of receptor editing. Thus, NF‐κB signaling in early B‐cell development is yet to be fully characterized. Therefore, we addressed the role of TAK1 in early B‐cell development. TAK1‐deficient mice showed significant reduction of BM Igλ‐positive B‐cell numbers without any alteration in the BCR editing. Furthermore, the expression of survival factor Bcl‐2 was reduced in TAK1‐deficient BM B cells as assessed by microarray and quantitative PCR analyses. Ex vivo over‐expression of exogenous Bcl‐2 enhanced the survival of TAK1‐deficient Igλ‐positive B cells. TAK1–IKK–NF‐κB signaling contributes to the survival of λ‐chain‐positive B cells through NF‐κB‐dependent anti‐apoptotic Bcl‐2 expression.     

SHIP represses mast cell activation and reveals that IgE alone triggers signaling pathways which enhance normal mast cell survival

The hemopoietic specific, Src homology 2-containing inositol 5' phosphatase (SHIP) hydrolyzes the phosphatidylinositol (PI)-3-kinase generated second messenger, PI-3,4,5-trisphosphate (PIP(3)), to PI-3,4-bisphosphate (PI-3,4-P(2)) in normal bone marrow derived mast cells (BMMCs). As a consequence, SHIP negatively regulates IgE+antigen (Ag)-induced degranulation as well as leukotriene and inflammatory cytokine production. Interestingly, in the absence of SHIP, BMMCs degranulate extensively with IgE alone, i.e. without Ag, suggesting that IgE alone is capable of stimulating signaling in normal BMMCs and that SHIP prevents this signaling from progressing to degranulation. To test this, we compared signaling events triggered by monomeric IgE versus IgE+Ag in normal BMMCs and found that multiple pathways are triggered by monomeric IgE alone and, while they are in general weaker than those stimulated by IgE+Ag, they are more prolonged. Moreover, while SHIP prevents this IgE-induced signalling from progressing to degranulation or leukotriene production it allows sufficient production of autocrine acting cytokines, in part by activation of NFkappaB, to enhance BMMC survival. Interestingly, the activation of NFkappaB and the level of cytokines produced are far higher with IgE than with IgE+Ag. Moreover, IgE alone maintains Bcl-X(L) levels and enhances the adhesion of BMMCs to fibronectin and this likely enhances their survival still further.